VirBR, a transcription regulator, promotes IncX3 plasmid transmission, and persistence of blaNDM-5 in zoonotic bacteria

IncX3 plasmids carrying the New Delhi metallo-β-lactamase-encoding gene, blaNDM-5, are rapidly spreading globally in both humans and animals. Given that carbapenems are listed on the WHO AWaRe watch group and are prohibited for use in animals, the drivers for the successful dissemination of Carbapenem-Resistant Enterobacterales (CRE) carrying blaNDM-5-IncX3 plasmids still remain unknown. We observe that E. coli carrying blaNDM-5-IncX3 can persist in chicken intestines either under the administration of amoxicillin, one of the largest veterinary β-lactams used in livestock, or without any antibiotic pressure. We therefore characterise the blaNDM-5-IncX3 plasmid and identify a transcription regulator, VirBR, that binds to the promoter of the regulator gene actX enhancing the transcription of Type IV secretion systems (T4SS); thereby, promoting conjugation of IncX3 plasmids, increasing pili adhesion capacity and enhancing the colonisation of blaNDM-5-IncX3 transconjugants in animal digestive tracts. Our mechanistic and in-vivo studies identify VirBR as a major factor in the successful spread of blaNDM-5-IncX3 across one-health AMR sectors. Furthermore, VirBR enhances the plasmid conjugation and T4SS expression by the presence of copper and zinc ions, thereby having profound ramifications on the use of universal animal feeds.


Fig. S1
Fig. S1 The proportion of blaNDM-5 of NDM-producing E. coli from human and animal in 2009-2014 and 2015-2022.a.The proportion of blaNDM-5 of NDM-producing E. coli from human and animal in 2009-2014.b.The proportion of blaNDM-5 of NDM-producing E. coli from human and animal in 2015-2022.

Fig. S2
Fig. S2 The proportion of blaNDM-5 of NDM-producing E. coli from animal and human in the world in 2009-2022.

Fig. S5
Fig. S5 The in vitro conjugation transfer efficiency of 3R (red bar) and 3RΔblaCTX-M-65 (blue bar) strains carrying the IncX3 plasmid under the pressure of either meropenem or amoxicillin.Data are means ± SEM; n = 3 biologically independent replicates.Groups were compared using two-tailed t-test.

Fig. S6 E
Fig. S6 E. coli carrying blaNDM-5-IncX3 plasmids can persist in chicken intestines.a. Bacterial loads of 5 different intestines of inoculated 3R group.b.Bacterial loads of 5 different intestines of inoculated 8R group.c. blaNDM-5positive strains load of large (red line) and small (green line) intestines of inoculated 8R group.d. blaNDM-5-positive strains load of large (red line) and small (green line) intestines of inoculated 3R group.

Fig. S7
Fig. S7 Core genome-based phylogenetic trees and MLST typing of the Non-inoculated NDM-positive E. coli strains.a. Core genome-based phylogenetic trees of the Non-inoculated NDM-positive E. coli strains.b.MLST typing of the Non-inoculated NDM-positive E. coli strains.c.The BRIG IncX3 plasmid comparative analysis of Non-inoculated NDM-positive E. coli strains.

Fig. S9
Fig. S9 Conjugation frequency of IncX3 plasmids and its derivatives in E. coli BW25113.Data are means ± SEM; n = 3 biologically independent replicates.One-way ANOVA was performed on values.

Fig. S12
Fig. S12 Fold changes of expression of genes in the condition of overexpression or deletion of virBR.a. Fold changes of expression of all genes in the condition of overexpression of virBR.Red plot represents up-expression of genes, and green plot represents down-expression of genes.b.Fold changes of expression of genes related to SOS response in the condition of overexpression or deletion of virBR.

Fig. S13 mRNA
Fig. S13 mRNA levels of T4SS genes in 3R and its derivatives.Data are means ± SEM; n = 3 biologically independent replicates.One-way ANOVA was performed on values.

Fig. S14
Fig. S14 Action site of virBR in the relaxsome and T4SS gene cluster.a. Schematic representation of the putative promoters in the relaxsome and T4SS gene cluster.Red arrows represent virBR, and green arrows represent the virBR downstream genes.b.Fluorescence intensity of GFP under the control of putative promoters in the absence (black bar) or presence (red bar) of virBR.Data are means ± SEM; n = 4 biologically independent replicates.Groups were compared using two-tailed t-test.

Fig. S15
Fig. S15 Co-transcription tests of actX and virB1.Primers located in the actX and virB1 coding sequence were used to amplified the Genomic DNA (1), RNA (2) and cDNA (3) were used as positive and negative controls respectively.

Fig. S17
Fig. S17 Strains load of feces of 3R and 3R-ΔactX group.a.The conjugation transfer of IncX3 plasmid and its derivatives ΔactX.Data are means ± SEM; n = 3 biologically independent replicates.One-way ANOVA was performed on values.b.Six mice of each group were euthanized and feces were taken to quantify the inoculated strains at 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21 after inoculated.Feces were coated Eosin-Methy Blue Agar Medium (containing 0.25 μg/ml meropenem) to count colony to do inoculated-strains quantitation.Data are means ± SEM; n = 6 biologically independent replicates.Groups were compared using two-tailed t-test.

Fig. S18 Effects of the deletion of virB1/ 2
Fig. S18 Effects of the deletion of virB1/2 on plasmid conjugation, plasmid invasion ability, cell adhesion ability and colonisation.a.The plasmid conjugation ability of 3R and its derivatives.Data are means ± SEM; n = 3 biologically independent replicates.One-way ANOVA was performed on values.b and c.Plasmid invasion ability of IncX3 plasmid in the presence or absence of virB1/2.d and e.The cell adhesion ability of 3R and its derivatives.